A great deal of interest has surrounded the use of monoclonal antibodies (mAbs) for the selective delivery of cytotoxic agents to tumor cells. MMAF (N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine) is an auristatin that is relatively non-toxic, yet is highly potent in activity when conjugated to internalizing mAbs. MMAF has a charged C-terminal phenylalanine residue that attenuates its cytotoxic activity compared to its neutral counterpart, MMAE; this difference is most likely due to impaired intracellular access. However, conjugating MMAF to internalizing antibodies, like AC10 or 1F6, via a protease cleavable linker resulted in conjugates that are >2000 fold more potent on antigen positive cells as compared to unconjugated drug. Active targeting with mAbs facilitates intracellular delivery of MMAF; once MMAF is released from the conjugate inside cells the drug, it is presumably trapped due to its reduced ability to cross cellular membranes, thus increasing its intracellular concentration and therefore the potency of the conjugate. Using cytotoxic drugs with impaired passive intracellular uptake can potentially lead to mAb-drug conjugates with reduced systemic toxicity. Indeed, non-specific cleavage of the linker in circulation would release a relatively non-toxic drug.
To expand and improve the auristatin class of drugs, and the corresponding antibody drug conjugates (ADCs), the C-terminal phenylalanine residue of MMAF has been replaced with carboxylic acid equivalents, such as phosphonophenylalanine (pPhe). This structural modification imparts unexpected properties to the resultant free drug and ADC. The recitation of any reference in this application is not an admission that the reference is prior art to this application.